Certain aggressive breast cancers that overexpress cyclin D can also express hyperactive Ras. Patients with corresponding tumors have poor outcomes associated with early onset of metastatic disease. Currently, these patients are treated in a similar manner to those with more favorable breast cancer biology and, hence, are not experiencing significant improvements in prognosis. Given the heterogeneity of aggressive breast cancer subtypes, the development of targeted treatment strategies is essential to improve patient survival rates. TGF- -regulated Smad3, a substrate of both cyclin D/CDK4 and Ras-activated JNK, exhibits a dichotomous tumor suppressant/oncogenic role in early and late stage breast cancer, respectively. The mechanism by which this oncogenic shift is exploited by cancer cells remains unclear. We hypothesize that the tumor suppressant and pro-metastatic effects of Smad3 are mediated by its site-specific phosphorylation, providing a link between TGF-, cyclin D/CDK4, RAS/JNK and the metastatic potential of breast cancer cells. The specific aims of this proposal will directly test this hypothesis as follows: Aim 1: Determine if CDK4 and JNK mediated phosphorylation of Smad3 regulates the transformation between TGF- induced tumor suppressive and pro-metastatic signaling in aggressive breast cancer cells. We will introduce Smad3 phosphorylation site mutants or treat cells with CDK4 or JNK inhibitors, and assess for Smad3-dependent markers of cell cycle control and EMT by mRNA and protein analysis. Aim 2: Elucidate the effect of CDK4 and JNK inhibition on tumor formation and metastasis in vitro, in 3D culture, and in a murine xenograft model of breast cancer. We will employ aggressive breast cancer cell lines transduced with CDK4/JNK phosphorylation site mutated Smad3 or treated with CDK4 or JNK inhibitor in migration and invasion assays, 3D tumor and murine xenograft models. Aim 3: Investigate the impact of CDK4- and JNK- mediated Smad3 phosphorylation on the activity of Smad3-regulated transcription factors in the context of an EMT focused signaling network, and determine key signature expression patterns of Smad3, cyclin D and Ras related proteins in aggressive breast cancer subtypes. We will employ a novel cell array to determine the effects of CDK4 and JNK inhibition on the activity of Smad3 associated EMT-promoting transcription factors that are implicated in several cancer-related signaling pathways, as well as use immunohistochemistry to assess the patterned expression of study proteins in human breast cancer tissues. Taken together, this work will contribute to the molecular staging of disease and facilitate discovery of novel therapeutic strategies that will expand treatment options for patients with aggressive breast cancer subtypes.